Alzheimer's disease (AD) is the most abundant form of dementia. It is characterized by slowly progressing neurodegeneration resulting in irrecoverable loss of cognitive functions. The constantly rising number of AD patients leads to financial and social burden for society. Thus, the development of prevention options as well as therapies is of great importance.
Aβ peptides play a crucial role in AD pathogenesis. They are generated from posttranslational processing of the amyloid precursor protein (APP), a type I transmembrane protein. Recent studies identified intracellular monomeric and oligomeric Aβ peptides as neurotoxic forms (oligomer hypothesis) whereof Aβ1-42 is the most pathogenic isoform. Synapses are mainly affected by Aβ neurotoxicity. It is known that mitochondria located in synapses exhibit dysfunctions already in the very early phase of AD pathogenesis. Hence, this study focused on mitochondria to characterize their significant functional alterations in the early phase of AD pathogenesis.
The human neuroblastoma cell line SH-SY5Y and the rat oligodendroglia cell line OLN-93 were used as model systems in this study. Cells were incubated with 2.0 µM disaggregated Aβ1-42 peptides for 24 h. The externally applied peptides were located intracellular. Then effects of Aβ1-42 on cell physiology, oxidative stress, energy metabolism, mitochondrial membranes and proteome were analyzed.
Disaggregation of Aβ1-42 peptides with trifluoroacetic acid resulted in monomers to heptamers. These peptides (labeled with fluorescent markers) were found in late endosomes after 5 h of incubation. After 16 h Aβ1-42 peptides were located in mitochondria. It appeared that mitochondria of OLN-93 cells incorporated higher amounts of Aβ1-42 peptides than SH-SY5Y cells. Due to the peptide's endosomal localization after a short incubation time it is postulated that its uptake is achieved by endocytosis.
AlamarBlue® assay and neutral red uptake assay were used to determine cell viability which was not significantly affected upon Aβ1-42 treatment. Externalization of phosphatidylserine is characteristic of early apoptotic cells. Phosphatidylserine presented to the extracellular matrix is recognized by Annexin V. However, late apoptotic cells as well as necrotic cells lose integrity of plasma membrane enabling propidium iodide to intercalate into DNA. There was no Aβ1-42 induced change in the number of apoptotic/necrotic cells for both cell lines which was detected by flow cytometry analysis after treatment with FITC Annexin V and propidium iodide.
Intracellular cleavage of the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate by esterases and oxidation by peroxidases in the presence of hydrogen peroxide results in the fluorescent 2',7'-dichlorofluorescein (DCF) that is used as a indicator for reactive oxygen species (ROS). Measuring the DCF fluorescence revealed a high increase (> 120%) in the cellular ROS amount in Aβ1-42 treated samples compared to untreated samples. Thus, Aβ1-42 leads to increased ROS generation and/or interference of ROS scavenging systems. ROS induce protein oxidation that may result in activity loss of affected proteins. Mitochondrial protein oxidation was determined by quantification of OxyBlots(TM) detecting protein carbonylation. Aβ1-42 treatment led to a slight increase of mitochondrial protein carbonylation that was interrelated to detections of cellular protein carbonylation by others.
Determination of specific activities of enzymes involved in oxidative phosphorylation (OxPhos complexes) and cellular ATP concentration was used to investigate the influence of Aβ1-42 peptides on energy metabolism. Photometric measurements with isolated mitochondria or cell lysates were applied to determine specific activities of OxPhos complexes. Both cell lines showed decreased activity of NADH-ubiquinone oxidoreductase (CI) but increased activity of cytochrome c oxidase (CIV) upon Aβ1-42 treatment. Aβ1-42 peptides did not influence activity of succinate dehydrogenase (CII) in OLN-93 cells, but decreased CII activity was found in SH-SY5Y cells. Activity loss of CI and CII implies further ROS generation. Increase of CIV activity is explained by enhanced protein amount and/or increased supercomplex formation. Cellular ATP concentration was identified via luciferase catalyzed and ATP mediated oxidative decarboxylation of D-luciferin. Measuring the emerging luminescence revealed a decrease in the cellular ATP concentration in Aβ1-42 treated compared to untreated samples. Thus, Aβ1-42 peptides cause reduced ATP synthesis and/or increased ATP consumption.
Aβ1-42 peptides also influenced mitochondrial membranes. Mitochondrial membrane potential (MMP) was analyzed via reduced MitoTrackers® by flow cytometry. In Aβ1-42 treated samples depolarization of the MMP was detected. It is explained by Aβ1-42 mediated loss in activity of CI and CII (demonstrated in this study). Aβ1-42 induced channel formation in mitochondrial membranes may also result in depolarization of the MMP. Steady-state anisotropy measurements of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene were used as indicator for "fluidity" of mitochondrial membranes. Aβ1-42 caused an increase of "fluidity" of mitochondrial membranes. The result of anisotropy measurements proves the intercalation of the peptide into the membrane.
For proteome analyses solubilized proteins obtained from isolated mitochondria were separated via two dimensional-blue-native/sodium dodecyl sulfate-polyacrylamid gel electrophoresis (2D-BN/SDS-PAGE). Protein subunits were identified by peptide mass fingerprint (PMF) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Quantification of protein subunits was executed on the basis of SYPRO® Ruby stained 2D-gels. A total of 41 proteins were identified for OLN-93 and 32 for SH-SY5Y, respectively. The huge advantage of 2D-BN/SDS-PAGE for the application in quantitative proteomics compared to the common method that uses denaturizing isoelectric focusing in the first dimension is the feasibility of separating hydrophobic membrane proteins as well as the preservation of protein-protein interactions. 14% of quantified subunits in OLN-93 samples were significantly influenced by Aβ1-42. Protein amounts of subunits α and β of F1FO ATP synthase (CV) monomer and dimer were reduced in Aβ1-42 treated OLN-93 samples. Thus, it is assumed that Aβ1-42 causes a reduction in ATP synthesis that explains the decreased cellular ATP concentration that was found in this study for Aβ1-42 treated OLN-93 samples. Furthermore, pyruvate kinase and a not identified protein showed decreased protein amounts upon Aβ1-42 treatment. In SH-SY5Y samples 30% of quantified subunits exhibited Aβ1-42 mediated alterations. Increased protein amounts of subunits α and β of CV dimer, subunit γ of CV monomer as well as of CII subunit SDHA were found in Aβ1-42 treated SH-SY5Y samples. These findings may be part of compensatory mechanisms to balance e.g. activity loss of CII (determined in this study) and CV. Additionally, four not identified proteins exhibited increased protein amounts. Decreased protein amounts were found for Hsp60 and three not identified proteins in Aβ1-42 treated SH-SY5Y samples.
This study revealed significant Aβ1-42 induced alterations of cell physiology and mitochondrial function. Findings may contribute to the development of prevention options as well as therapies since they provide information about the early phase of AD pathogenesis.